In recent years recombinant protein/enzyme production for use in industrial processes has become more and more important and it is expected that soon many industrial processes will involve recombinant technologies. Currently, bioactive peptides and proteins are used as curative agents in a variety of diseases such as diabetes (insulin), viral infections and leukemia (interferon), diseases of the immune system (interleukins), and red blood cell deficiencies (erythropoietin) to name a few. Additionally, large quantities of proteins and peptides are needed for various industrial applications including, for example, the pulp and paper industries, textiles, food industries, personal care and cosmetics industries, sugar refining, wastewater treatment, production of alcoholic beverages and as catalysts for the generation of new pharmaceuticals.
However, the expression of recombinant peptides and proteins is still limited, as large efforts are required in order to obtain the desired peptides and proteins with a native fold, in high amounts and high purity.
Generally, product purification is expensive and especially the final step to 100% purity tends to increase the costs exponentially because proteins with similar characteristics are difficult to separate from one another (Hacking, A. J. (1986) Economic aspects of biotechnology, Cambridge University Press).
In many cases it is useful to express a protein or peptide in insoluble form, particularly when the peptide of interest (PeOI) or protein of interest (PrOI) is rather short, normally soluble, and/or subject to proteolytic degradation within the host cell. Production of the peptide in insoluble form both facilitates simple recovery and protects the peptide from the undesirable proteolytic degradation. One means to produce the peptide in insoluble form is to recombinantly produce the peptide as part of an insoluble fusion peptide/protein by including in the fusion peptide at least one solubility tag (i.e., an inclusion body (IB) tag) that induces IB formation. Typically, the fusion protein is designed to include at least one cleavable peptide linker so that the PeOI or PrOI can be subsequently recovered from the fusion protein. The fusion protein may be designed to include a plurality of IB-tags, cleavable peptide linkers, and regions encoding the PeOI or PrOI.
Fusion proteins comprising a peptide tag that facilitate the expression of insoluble proteins are well known in the art. Typically, the tag portion of the chimeric or fusion protein is large, increasing the likelihood that the fusion protein will be insoluble. Example of large peptides typically used include, but are not limited to chloramphenicol acetyltransferase (Dykes et al., (1988) Eur. J. Biochem., 174:411), β-galactosidase (Schellenberger et al., (1993) Int. J. Peptide Protein Res., 41:326; Shen et al., (1984) Proc. Nat. Acad. Sci. USA 281:4627; and Kempe et al., (1985) Gene, 39:239), glutathione-S-transferase (Ray et al., (1993) Bio/Technology, 11:64 and Hancock et al. (WO94/04688)), the N-terminus of L-ribulokinase (U.S. Pat. No. 5,206,154 and Lai et al., (1993) Antimicrob. Agents & Chemo.), 37:1614, bacteriophage T4 gp55 protein (Gramm et al., (1994) Bio/Technology, 12:1017), bacterial ketosteroid isomerase protein (Kuliopulos et al., (1994) J Am. Chem. Soc. 116:4599 and in U.S. Pat. No. 5,648,244), ubiquitin (Pilon et al., (1997) Biotechnol. Prog., 13:374-79), bovine prochymosin (Haught et al., (1998) Biotechnol. Bioengineer. 57:55-61), and bactericidal/permeability-increasing protein (“BP1”; Better, M. D. and Gavit, P D., U.S. Pat. No. 6,242,219). The art is replete with specific examples of this technology, see for example U.S. Pat. No. 6,037,145, teaching a tag that protects the expressed chimeric protein from a specific protease; U.S. Pat. No. 5,648,244, teaching the synthesis of a fusion protein having a tag and a cleavable linker for facile purification of the desired protein; and U.S. Pat. Nos. 5,215,896; 5,302,526; 5,330,902; and U.S. Patent Application Publication No. 2005/221444, describing fusion tags containing amino acid compositions specifically designed to increase insolubility of the chimeric protein or peptide.
However, the methods known in the art do not provide any solution to refold the PeOI or PrOI. Thus, there is still need in the art for methods that allow improved production of a recombinant PeOI and PrOI.
The present inventors found that methods comprising nucleic acid sequences comprising hemolysin A (HlyA) or lipase A (LipA) gene fragments overcome the above need in the art. Both genes are part of a Type 1 secretion system (TISS), which mostly occur in Gram-negative bacteria and export their cognate substrates in a single step from the cytosol to the extracellular medium without the formation of periplasmic substrate intermediates. Among the family of TISS the Hly TISS described by Bakkes et al. involving HlyA as transport substrate is of particular interest, as it carries so-called GG repeats with the consensus sequence GGxGxDxUx (SEQ ID NO: 67(x: any amino acid residue, U: large, hydrophobic amino acid residue) (Ostolaza, H. et al., (1995) Eur J Biochem 228, 39-44). These GG repeats bind Ca2+ ions with high affinity. This binding event happens after the secretion of the TISS allocrite to the exterior, where the Ca2+ concentration is high (up to the mM range) compared to the Ca2+ concentration inside the cells (high nM). Ca2+ binding to the GG repeats catalyzes the folding of the allocrites into the native, active conformation and Ca2+ ions act as a folding helper/chaperone (Jumpertz, T. et al., Microbiology 156, 2495-2505, doi:mic.0.038562-0 [pii]). Further components of the HlyA TISS of E.coli are the inner membrane protein HlyB, which is an ATP binding cassette (ABC) transporter, the outer membrane protein (OMP) TolC and the membrane fusion protein (MFP) HlYD in the inner membrane.